Two antioxidative lactobacilli strains as promising probiotics

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Abstract

Two antioxidative strains tentatively identified as Lactobacillus fermentum, E-3 and E-18, were isolated from intestinal microflora of a healthy child. Survival time of these strains in the presence of reactive oxygen species (ROS), like hydrogen peroxide, superoxide anions and hydroxyl radicals, was significantly increased compared with a non-antioxidative strain, and also was quite similar to a highly ROS resistant strain of Salmonella typhimurium. E-3 and E-8 contain a remarkable level of glutathione, express Mn-SOD, which is important for the prevention of lipid peroxidation, and secrete hydrogen peroxide. Their significant antimicrobial activity combined with antioxidative properties may serve as defensive principles in the intestinal microbial ecosystem and overcome exo- and endogenous oxidative stress.

Introduction

The increasing interest in healthy diet is stimulating the innovative development of novel products by the food industry, and intestinal Lactobacillus species are particularly in focus (Vaughan et al., 1999). During the past decade, oxidative stress and antioxidative potency have been revealed as the key points in molecular regulation of cellular stress responses (Demple et al., 1999). Oxidative stress occurs when abnormally high levels of reactive oxygen species (ROS) are generated, resulting in DNA, protein and lipid damage. Both eukaryotic and aerobic prokaryotic organisms have developed an overall antioxidative defense system to mitigate the damaging effects of ROS. The important components of the cellular defense system are reduced glutathione (GSH) and antioxidative enzymes like superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase (CAT) Frei et al., 1988, Halliwell et al., 1992, Vervaart and Knight, 1996, Miller and Britigan, 1997.

In some human microbiota, Lactobacillus, Streptococcus spp., lacking cytochromes and catalase, the flavoproteins (e.g. NADH-oxidase) make oxygen consumption possible, and give rise to an increase of the content of superoxide anions and hydrogen peroxide, which are highly toxic to several microorganisms and are considered to have an impact on bacterial antagonism in microbial ecology Dahiya and Speck, 1968, Eschenbach et al., 1989. Concerning Lactobacillus spp., it has been concluded that their antimicrobial effect is also expressed via ROS (hydrogen peroxide, superoxide and hydroxyl radicals) which may have a selective influence on the intestinal microbiota, similar to that described for oral streptococci (Garcia-Mendoza et al., 1993).

Lactobacillus spp. are important members of the healthy human microbiota Mikelsaar et al., 1984, Lidbeck and Nord, 1993, Mikelsaar et al., 1998. Lactic acid bacteria and bifidobacteria are considered to have several beneficial physiological effects, such as antimicrobial activity, enhancing of immune potency and antitumorigenic activities Fuller, 1991, Salminen et al., 1998. It has been shown that some lactobacilli possess antioxidative activity, and are able to decrease the risk of accumulation of ROS during the ingestion of food Kaizu et al., 1993, Peuhkuri et al., 1996. Lactic acid bacteria are able to degrade the superoxide anion and hydrogen peroxide Ahotupa et al., 1996, Korpela et al., 1997. However, the type of superoxide dismutase (SOD) expressed in antioxidative strains has not been assessed. It still remains open if the antioxidative potency of strains is associated with their survival in different environment.

In this study, we report that strains E-3 and E-18, tentatively identified as Lactobacillus fermentum with substantial antioxidative activity, express Mn-SOD (manganese superoxide dismutase) and have significantly increased resistance to several ROS, like hydrogen peroxide, superoxide and hydroxyl radicals.

Section snippets

Strains of microorganisms

Strains of lactobacilli and Salmonella typhimurium were used in this study. Lactobacilli (E-3 and E-18) were isolated from the faecal sample of an Estonian child and E-338-1-1 was isolated from another healthy Estonian 1-year-old child (Sepp et al., 1997). Isolation of lactobacilli on de Man–Rogosa–Sharpe agar (MRS agar; CM 361, Oxoid Basingstoke, Hampshire, UK) was followed by the tentative identification to the species level by the API 50 CHL kit and API LAB Plus software, version 4.0

Results

Two strains of L. fermentum, E-3 and E-18, had significant antioxidative capacity established by using LA-test and TAS-test (Table 1). We also examined several parameters of these antioxidative strains (E-3 and E-18) including their survival in the presence of ROS in comparison with the survival of non-antioxidative strain L. fermentum E-338-1-1 (Table 1 and Fig. 3) and with a highly ROS resistant strain of S. typhimurium.

Discussion

Two antioxidative strains of L. fermentum were isolated from the intestinal microflora of a healthy child. In this study, we examined the resistance of highly antioxidative strains of L. fermentum, named as E-3 and E-18, to the different unhealthy milieu of ROS. We compared the survival of E-3, E-18 both with the non-antioxidative strain of L. fermentum E-338-1-1 and with S. typhimurium.

Our major findings were as follows: the strains E-3 and E-18 (compared with the non-antioxidative strain

Acknowledgements

We thank Mrs. Mai Laanes for excellent technical assistance. This work was supported by funding No. 3358 and 0418 from the Estonian Ministry of Education.

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